β-Lapachone Ameliorates Lipotoxic Cardiomyopathy in Acyl CoA Synthase Transgenic Mice

<div><p>Lipotoxic cardiomyopathy is caused by myocardial lipid accumulation and often occurs in patients with diabetes and obesity. This study investigated the effects of β-lapachone (β-lap), a natural compound that activates Sirt1 through elevation of the intracellular NAD⁺ level, on acyl CoA synthase (ACS) transgenic (Tg) mice, which have lipotoxic cardiomyopathy. Oral administration of β-lap to ACS Tg mice significantly attenuated heart failure and inhibited myocardial accumulation of triacylglycerol. Electron microscopy and measurement of mitochondrial complex II protein and mitochondrial DNA revealed that administration of β-lap restored mitochondrial integrity and biogenesis in ACS Tg hearts. Accordingly, β-lap administration significantly increased the expression of genes associated with mitochondrial biogenesis and fatty acid metabolism that were down-regulated in ACS Tg hearts. β-lap also restored the activities of Sirt1 and AMP-activated protein kinase (AMPK), the two key regulators of metabolism, which were suppressed in ACS Tg hearts. In H9C2 cells, β-lap-mediated elevation of AMPK activity was retarded when the level of Sirt1 was reduced by transfection of siRNA against Sirt1. Taken together, these results indicate that β-lap exerts cardioprotective effects against cardiac lipotoxicity through the activation of Sirt1 and AMPK. β-lap may be a novel therapeutic agent for the treatment of lipotoxic cardiomyopathy.</p></div

β-Lap preserves mitochondrial structure and functions in ACS Tg mice.

<p><b>A</b>. Electron microscopy images of mitochondrial structure and quantification of the mitochondrial surface area in sections of hearts from Wt and ACS Tg mice administered either vehicle (Veh) or β-lap (βL). Scale bar is 1 µm. <b>B</b>. Western blot analysis of Complex II protein level. Mice heart lysates (50 µg) were subjected to western blot analysis, and band density was quantified using NIH Image J software. GAPDH was used as a loading control. <b>C</b>. qRT-PCR analysis of mt-Co1 and mt-Cyt b transcript levels in mouse hearts. <b>D</b>. qRT-PCR analysis of genes involved in mitochondrial biogenesis (NRF-1, PPARα, ERRαα and PGC-1β) and FA metabolism (MCAD, PDK4, GPAT, CPT1-β, UCP2, and ATP6i). n = 4−6 per group. Significance was measured via two-way ANOVA. *<i>p</i><0.05, **<i>p</i><0.001.</p

Echocardiographic parameters.

<p>Wt, wild-type mice; Tg, ACS overexpressing transgenic mice; Veh, vehicle; βL, β-lapachone; IVSTd, Interventricular septum in diastole; LVIDd, Left ventricular internal dimension in diastole; LVPWd, Left ventricular posterior wall thickness in diastole; IVSTs, Interventricular septum in systole; LVIDs, Left ventricular internal dimension in systole; LVPWs, Left ventricular posterior wall thickness in systole; EF, Ejection fraction; FS, Fractional shortening. Significance was measured via two-way ANOVA.</p><p>**<i>p</i><0.001 vs Wt,Veh.</p>≠<p><i>p</i><0.05 vs Tg,Veh.</p>≠ ≠<p><i>p</i><0.001 vs Tg,Veh.</p

β-Lap prevents lipid accumulation in the heart of ACS Tg mice.

<p>Triglyceride contents in hearts from Wt and ACS Tg mice administered either vehicle (Veh) or β-lap (βL). n = 7−8 per group. Significance was measured via two-way ANOVA. *<i>p</i><0.05, **<i>p</i><0.001.</p

β-Lap attenuates cardiac hypertrophy and fibrosis in ACS Tg mice.

<p><b>A</b>. Cross-sections of the hearts and assessment of heart weight/body weight (HW/BW) ratios in Wt and ACS Tg mice administered either vehicle (Veh) or β-lap (βL). Scale bar is 1 mm. <b>B</b>. Higher magnification images of the heart sections. Cell surface area (CSA) of individual cardiomyocytes was measured using the AnalySIS image analysis program. Scale bar is 50 µm. <b>C</b>. Trichrome staining of histological sections. Fibrotic areas were quantified using the AnalySIS image analyzer on histological sections. Scale bar is 100 µm. <b>D</b>. qRT-PCR analysis of collagen I and TGF- β1 transcript levels in mouse hearts. n = 4−6 per group. Significance was measured via two-way ANOVA. *<i>p</i><0.05, **<i>p</i><0.001.</p

β-Lap preserves cardiac functions in ACS Tg mice.

<p><b>A</b>. Two-dimensional guided M-mode echocardiographic images were obtained from Wt and ACS Tg mice administered either vehicle (Veh) or β-lap (βL). <b>B</b>. Quantification of cardiac structure and function assessed by echocardiography. The leftventricular internal dimension at diastole (LVIDd), leftventricular internal dimension at systole (LVIDs), and fractional shortening (FS) values are shown. n = 9−12 per group. Significance was measured via two-way ANOVA. *<i>p</i><0.05, **<i>p</i><0.001.</p

β-Lap stimulates the AMPK signaling pathway through activation of Sirt1.

<p><b>A</b>. Mouse hearts lysates (1 mg) were immunoprecipitated with an anti-acetyl lysine antibody. The precipitates and whole extracts (50 µg) were analyzed by western blotting. The levels of the acetylated forms of LKB1, FOXO, p53, and PARP1 were estimated by measuring band densities using NIH Image J software. GAPDH was used as a loading control. <b>B</b>. Western blot analysis of the protein levels of ACS, p-AMPK, AMPK, p-ACC, and ACC. Mouse heart lysates (50 µg) were subjected to western blot analysis, and levels of phosphorylated AMPK and ACC were estimated by measuring band densities using NIH Image J software. GAPDH was used as a loading control. n = 3−4 per group. Significance was measured via two-way ANOVA. *<i>p</i><0.05, **<i>p</i><0.001.</p

Science and society: in the sixteenth and seventeenth centuries

eIF2α phosphorylation in hepatocytes is dispensable for survival of adult mice. (a) Diagram depicting the four genotypes of mice used in these experiments. S/A and A/A represent heterozygous and homozygous eIF2α Ser51Ala (*) mutation(s) in exon 2 of one eIF2α allele and both eIF2α alleles, respectively. fTg/0 represents the floxed wild type (WT) eIF2α transgene driven by the CMV enhancer and chicken β-actin promoter (Enh-Pro). The loxP sequences (black arrowheads) allow excision of the WT eIF2α floxed transgene (fTg) and expression of EGFP, an indicator of recombination. CRE Hep /0 represents the Cre recombinase transgene driven by the promoter (Alfp) of Alb1 (encoding albumin) and the enhancer of Afp (encoding alpha-fetoprotein). (b) Efficiency of deletion of the fTg in liver tissues. Results from quantitative RT-PCR analyses of transgenic and total eIF2α mRNAs are shown. Data are means ± SEM (n = 4 ~ 5 mice per group); ### p < 0.001; Cont. vs A/A Hep . (c) Western blot analysis of eIF2α protein expression driven by the fTg in liver tissues. To quantity expression of eIF2α, blots were incubated with anti-eIF2α antibody followed by IRDye-800 goat anti-rabbit IgG (LI-COR). Membranes were scanned on an Odyssey scanner (LI-COR) (lower two panels in left panels) and quantified with the Odyssey Software package. (d) Western blot analysis of liver lysates in Cont. and A/A Hep mice at the indicated times after Tm injection. Cont. mice and A/A Liv mice were injected with vehicle or tunicamycin (Tm, 1 mg/kg). (e) Body weight measurements of fTg -deleted A/A Liv mice. At the weeks, body weight was measured in both male and female mice. Data are means ± SEM (n = 6-14 mice per group). (PDF 1987 kb

Additional file 2: Table S2. of eIF2Îą phosphorylation is required to prevent hepatocyte death and liver fibrosis in mice challenged with a high fructose diet

PCR primers. (XLS 40 kb

Transcriptional regulation of bile acid enzyme genes by <i>Crebh.</i>

<p>(<b>A</b>) Mice (n = 5) or primary human hepatocytes (n = 3) were infected with indicated adenoviruses for 96 hrs. Liver tissues were obtained and protein and total RNA was extracted for western blot and qPCR analyses, respectively. *<i>p</i><0.05 vs. Ad-GFP group. (<b>B</b>) HepG2 cells were co-transfected with CREBH-N and different CYP7A1-Luc and CYP27A1-Luc promoter constructs, and luciferase assay was performed. (<b>C–D</b>) HepG2 cells were transfected with wild type (wt) or CREBH-mutant (mut) constructs of CYP7A1-Luc or CYP27A1-Luc followed by 2-AG-ether treatment for 12 hrs and luciferase assay was performed (D) or immunoprecipitation of HepG2 chromatin from cells exposed to DMSO (control) or 2-AG-ether was performed with IgG or Crebh antibody (E). Promoter regions were amplified by PCR, as depicted. Percentage of DNA immunoprecipitated with Crebh antibody relative to input chromatin was quantified by qPCR. *<i>p</i><0.05 vs. control. Data represents mean ± SE.</p